THE COMPOSITION AND ANTIMICROBIAL ACTIVITY OF LEAF ESSENTIAL OILS OF SELECTED AGATHOSMA SPECIES (RUTACEAE) Carla Fourie (Student number: 0111602D) A research report submitted to the faculty o f Health Sciences, University o f the Witwatersrand, Johannesburg, in partial fulfillment of the requirements for the degree o f Master o f Science in Medicine (Pharmaceutical Affairs). Johannesburg, 2003
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THE COMPOSITION AND ANTIMICROBIAL
ACTIVITY OF LEAF ESSENTIAL OILS OF SELECTED
AGATHOSMA SPECIES (RUTACEAE)
Carla Fourie
(Student number: 0111602D)
A research report submitted to the faculty o f Health Sciences, University o f the
Witwatersrand, Johannesburg, in partial fulfillm ent o f the requirements fo r the
degree o f Master o f Science in Medicine (Pharmaceutical Affairs).
Johannesburg, 2003
Declaration
I, Carla Fourie declare that this research report is my own work. It is being submitted for the degree o f MSc (Med) Pharmaceutical Affairs in the University of the Witwatersrand, Johannesburg. It has not been submitted before for any degree or examination at this or any other University.
it . .ih
day of N w jp fth r.... , 2003
2
Acknowledgements
I would like to thank the following:
My supervisors Dr A. Viljoen and Ms S. van Vuuren.
My husband, Mome.
Prof Ba§er, Drs Bettil Demirci and Temel Ozek from the Medicinal and Aromatic Plant and Drug Research Centre, University of Anadolu, Turkey for the hospitality and efficient help with the GC-MS analysis during my visit to the Research Centre.
Ms Janine Victor of the National Botanical Institute and Mr Trinder-Smith (Bolus Herbarium) for assisting in the collecting and identification o f the various plant specimens.
The National Research Foundation, University o f the Witwatersrand Research Committee and Faculty of Health Sciences Research Endowment fund for financial support.
3
Table of contents
List of figures 6
List of tables 8
Abstract 9
1. Introduction:
1.1 History 10
1.2 Essential oils 11
1.3 Microbiological activity o f essential oils 12
1.4 The Rutaceae 14
1.5 The genus Agathosma 15
1.6 Previous research on Agathosma 16
2. Material and methods
2.1 Collection o f plant material 20
2.1.1 Extraction o f essential oils 20
2.2 Antimicrobial testing 21
2.2.1 Test organisms 21
2.2.2 Disc diffusion assay 21
2.2.3 MIC/microplate bioassay method 23
2.3 Analytical chemistry 25
2.3.1 Thin-layer chromatography (TLC) 25
2.3.2 Gas chromatograph-Mass Spectrometry (GC-MS) 25
Escherichia coli (ATCC 8739), Bacillus cereus (ATCC 11778) and Salmonella
typhimurium (clinical strain).
Fungal test organisms were as follow: Candida albicans (ATCC 10231),
Cryptococcus neoformans (ATCC 90112) and Aspergillus niger (clinical strain).
2.2.2 Disc diffusion assay
Four large Mueller-Hinton (Oxoid) agar plates were prepared containing 100 ml of
agar and 100 ml o f overlayed agar. Overnight broth cultures o f the following bacteria;
Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli and Enterococcus
faecalis, with an inocculum size lxlO 6 was used to seed the agar. The bacterial spore
suspensions were incorporated into the top agar layer. Smaller agar plates (Figure 3)
were prepared for Salmonella typhimurium and Bacillus cereus. The smaller round
petri dishes contained 15 ml of agar and 15 ml o f agar seeded with inoculum.
Aseptic techniques were used to saturate discs (Figure 4) with essential oils and then
placed onto the seeded agar plates. A disc containing Neomycin 30 pg, (Oxoid) was
used as a positive bacterial control. The screening plates were refrigerated for 1 hour
to allow pre-diffusion and then incubated at 37 °C for 24 hours, after which zones of
inhibition were measured.
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Figure 4: Aseptic transfer of discs with Agathosma essential oil onto seeded agar plate.
Figure 5: Preparing the MIC plate by performing doubling dilutions o f Agathosma oil.
Figure 2: Charging the stills with plant material. Figure 3: Preparation of plates for discdiffusion method by pouring base layer and proceeding with inoculated top layer.
A second series o f fungal test organisms namely Candida albicans, Cryptococcus
neoformans and Aspergillus niger were also studied. A disc containing Nystatin (100
lp., Oxoid) was used as a positive control. Tryptone Soya (Oxoid) agar was used for
the fungi and the same process as described above was followed to prepare the agar
and the saturated disc.
2.2.3 MIC/microplate bioassay method
After antimicrobial activity was recorded by the disc diffusion assay, the minimum
inhibitory concentrations (MIC) o f the most active oils were determined by using the
/i-iodonitrotetrazolium violet (INT) microplate method. A fixed bacterial culture
yielding an inoculum size o f lxlO6 was added to all wells. Further more, this method
involves dilution of the essential oils in microplate test conditions. An essential oil
concentration o f 32 mg/ml was prepared in the first row o f wells in the microplate
(Figure 5). Serial dilutions were performed (Figure 6). The addition o f the oils to the
microplate as well as the serial dilutions took place under laminar airflow to minimize
contamination.
Minimum inhibitory concentration (MIC) o f the following essential oils; A. ovata
(Gamka and Anysberg), A. capensis (Gamka) and A. recurvifolia, were determined for
Staphylococcus aureus, Enterococcus faecalis and Escherichia coli.
These oils and bacteria were chosen using the screening results as a guide to their
positive antimicrobial properties. Overnight broth cultures o f these organisms were
prepared as for the disc diffusion assay. A fixed bacterial culture yielding an inoculum
size o f lxlO6 was added to all wells. The microplate (Figure 5) was incubated for 24
hours at 37°C. Forty microliters o f INT solution, with a concentration o f 0.2 mg/ml,
was added to all wells after incubation. INT binds in a complex manner with the
DNA in bacterial cells. The INT solution is used as a bacterial growth indicator. The
pink colour change was observed after 30 minutes, 2 hours and 24 hours. The
minimum inhibitory concentration, o f each sample o f hydrodistilled oil, was
calculated. Minimum inhibitory concentrations were determined presumptively as the
first well, in ascending order, which did not produce a colour change.
23
1 2 3 4 5 6 7 8 9 1011 12AO o o o o o o o o o o oBO o o o o o o o o o o oCO o o o o o o o o o o oDO o o o o o o o o o o oeO o o o o o o o o o o oFO o o o o o o o o o o oGO o o o o o o o o o o oHO o o o o o o o o o o o
Column 1: A. capensis (Gamka) and Escherichia coli Column 2: A. capensis (Gamka) and Staphylococcus aureus Column 3: A. capensis (Gamka) and Enterococcus faecalis Column 4: A. ovata (Gamka) and Escherichia coli Column 5: A. ovata (Gamka) and Staphylococcus aureus Column 6: A. ovata (Gamka) and Enterococcus faecalis Column 7: A. ovata (Anysberg) and Escherichia coli Column 8: A. ovata (Anysberg) and Staphylococcus aureus Column 9: A. ovata (Anysberg) and Enterococcus faecalis Column 10: A. recurvifolia and Escherichia coli Column 11: A. recurvifolia and Staphylococcus aureus Column 12: A. recurvifolia and Enterococcus faecalis
Row A: Concentration o f essential oil = 32 mg/ml Row B: Concentration o f essential oil = 16 mg/ml Row C: Concentration o f essential oil = 8 mg/ml Row D: Concentration o f essential oil = 4 mg/ml Row E: Concentration o f essential oil = 2 mg/ml Row F: Concentration o f essential oil = 1 mg/ml Row G: Concentration o f essential oil = 0.5 mg/ml
Row H: Concentration o f essential oil = 0.25 mg/ml
Figure 6: Serial dilutions o f the essential oils o f A. capensis (Gamka), A. ovata (Gamka), A. ovata (Anysberg) and A. recurvifolia.
2.3 Analytical chemistry
2.3.1 Thin layer chromatography (TLC)
Silica thin layer plates were used together with toluene-ethyl acetate (93:7) as mobile
phase. Detection was made possible with the use o f spray reagents namely vanillin-
sulphuric acid and anisaldehyde-sulphuric acid.
After development, the TLC plates were air-dried and sprayed with one o f the
reagents. Colour development took place after a short heating period.
2.3.2 Gas chromatograph-Mass Spectrometry (GC-MS)
Essential oils were analyzed using the following operating conditions: Column: HP-
Innowax (60 m x 0.25 mm id., 0.25 jum film thickness), Temperatures: injection port
250 °C, column 60 °C for 10 min., 4 °C / min. to 220 °C, 220 °C for 10 min., 1 °C /
min. to 240 °C (total = 80 min.). Helium as carrier gas.
0.9 ill o f hexane with 0.1 /zl o f the essential oil was injected. Identification took place
with the use o f TBAM’s database libraries by matching both retention indices and
mass spectral fragmentation patterns. This part o f the project was completed by
myself at the Medicinal and Aromatic Plant and Drug Research Centre, Anadolu
University, Turkey.
2.3.3 TLC bioautographic assay
The hydrodistilled oil o f Agathosma zwartbergensis was chosen for the TLC
bioautographic assay. A Bacillus cereus spore suspension with an inoculum size of
lxlO6 was incorporated into Mueller Hinton agar. TLC plates o f the essential oil and
of a standard o f the main compound were developed and placed directly onto the
prepared agar plate. The TLC plates were developed by using toluene-ethyl acetate
(93:7) as the mobile phase and vanillin-sulphuric acid spray reagent to detect the
compounds o f the oil. The TLC overlay-agar plate was incubated for 24 hours.
25
3. Monographs of Agathosma species studied
Agathosma arida P.A. Bean
1. Botanical description
Single-stemmed, rounded shrublet to 40cm, sweetly herb-scented. Flowers in
terminal clusters, pink or violet. Fruits: 3-chambered. Ovary: usually 3-lobed.
2. Distribution
Gravelly loam, karoo-fynbos ecotone. This species is restricted to the Little Karoo,
specifically the northern slopes of Langeberg and Outeniqua Mountains (Goldblatt
and Manning, 2000).
Figure 7: Geographical distribution of A. arida.
3. Essential oil composition
Figure 8: Gas chromatography profile o f A. arida.
T o ta l 96.95RRP-retention indices calculated against n-alkanes
Various GC-MS libraries could not identify the major compound o f A. mundtii. This
compound had a base peak of 69 and a molecular mass o f 172. 76.95% (61
compounds) o f the essential oil composition was identified with the following six
monoterpenes as major compounds: linalool (19 %), myrcene (10 %), terpinen-4-ol
(9.62%), (Z)-P-ocimene (5.84%), sabinene (4.79%) and |3-pinene (4.62%).
terpinen-4-ol myrcene linalool
(B-pinene (Z)-p-ocimene sabinene
Figure 20: Chemical structures of the major compounds identified in A. mundtii
essential oil.
39
Agathosma ovalifolia Pillans
1. Botanical description
Single-stemmed, rounded shrub to 1.5m, acrid or spice-scented. Flowers in lax
terminal clusters white, red-dotted. Fruits: 2-chambered. Ovary: usually 1- or 2-
lobed.
2. Distribution
Rocky quartzitic upper slopes. This species is distributed from the Swartberg
Mountains to Willowmore (Goldblatt and Manning, 2000).
Figure 21: Geographical distribution of A. ovalifolia.
3. Essential oil composition
, ' V , . ............. .. , ' P * ,n , r ^ i —,10.00 15-00 20.00 25.00 30.00 35.00 -40-00 -45.00 50.00 55.00 60. OC
Figure 22: Gas chromatography profile of A. ovalifolia.
40
Table 6: GC-MS results of A. ovalifolia.R R I* R e ten tio n tim e A rea p ercen ta g e C om p ou n d1032 8.58 1.55 a-pinene1035 8.70 0.20 a-thujene (not integrated)1118 12.18 1.18 P-pinene1132 12.87 6 .3 6 sa b in en e1159 14.13 0.03 5-3-carene
Figure 36: Chemical structures o f the major compounds identified in A. zwartbergensis essential oil.
54
4. Results and Discussion
4.1 Antimicrobial activityThe antimicrobial results are summarized in Tables 11, 12 and 13. Antibacterial and
antifungul screening was done followed by the determination o f the minimum
inhibitory concentration (MIC) for selected oil samples showing positive
antimicrobial activity in the disc diffusion assay.
Table 11 shows the disc diffusion antibacterial screening results o f all the Agathosma
species studied. The results were taken after 24 hours of incubation. Zones of
inhibition were measured in millimeters from the edge of the disc containing the
essential oil. All the species studied showed a variable degree o f antibacterial activity.
Agathosma capensis (Mossel Bay) showed activity against E. coli, E. faecalis and S.
aureus. Figure 37 shows a broadscreening of E. coli on all Agathosma oils.
Agathosma lanata and A. zwartbergensis only showed antibacterial activity against B.
cereus. Figure 38 shows the zones of inhibition on selected oil samples (34 - A.
capensis (Gamka); 3 6 - A. ovata (Gamka); 38 -A . zwartbergensis; 40 -A . ovalifolia)
for B. cereus. The A. capensis and the A. ovata samples that were both collected from
the Gamka Mountains in the Cape and Agathosma serpyllacea showed activity against
all test organisms except P. aeruginosa. The A. capensis (Rooiberg) sample showed
little activity against E. coli, S. typhimurium and S. aureus', A. recurvifolia showed
activity against E. coli, E. faecalis and S. aureus', A. ovalifolia showed activity against
E. faecalis and to a lesser extend S. aureus and A. arida showed activity against S.
typhimurium and B. cereus. Agathosma mundtii showed activity against E. faecalis,
S. typhimurium, S. aureus and some initial activity against B. cereus. The activity of
A. mundtii against B. cereus was difficult to determine as initial zones were detected
after 24 hours but regrowth was noted after 48 hours, thus indicating that the essential
oil probably evaporated.
The A. ovata (Anysberg) sample has minimal broad-spectrum antibacterial activity
and was the only species that showed activity against P. aeruginosa.
The largest zone o f inhibition was 7.0 mm from the disc and was the result of the
essential oil o f A. recurvifolia's activity against E. faecalis. Neomycin 30 pg, (Oxoid)
55
was used as a positive control and measured 3.0 mm from the disc, therefore the
inhibition o f the essential oil o f A. recurvifolia was greater than that o f the control.
The antifungal results were similar to the antibacterial results. A small range o f fungi
were however tested. Table 12 shows the antifungal screening results o f all
Agathosma species studied. All the species showed activity against C. neoformans,
with A. arida exhibiting the largest zone (3.0 mm) o f inhibition. No activity was
noted for both C. albicans and A. niger for A. arida. The following species showed
antifungal activity against C. albicans'. A. ovata (Gamka), A. zwartbergensis, A.
ovalifolia, A. recurvifolia and A. serpyllacea. Agathosma ovalifolia was the only
species showing some minimal activity against A. niger.
The minimum inhibitory concentration (MIC) o f the essential oils on the test bacteria
was determined by using the p-iodonitrotetrazolium violet (INT) microplate method.
The MIC o f A. ovata (Gamka and Anysberg samples), A. recurvifolia and A. capensis
(Gamka sample) were determined on E. coli, S. aureus and E. faecalis. The results as
summarized in Table 13 and in Figure 39. These results reflect that the concentration
o f A. capensis (Gamka) oil needed to inhibit the growth o f E. coli is 16 mg/ml, S.
aureus is 32 mg/ml and E. faecalis is 32 mg/ml. The concentration o f A. ovata
(Gamka) oil needed to inhibit the growth oiE . coli is 16 mg/ml, S. aureus is 8 mg/ml
and E. faecalis is 16 mg/ml. The concentration o f A. ovata (Anysberg) oil needed to
inhibit the growth o f E. coli is 8 mg/ml, S. aureus is 8 mg/ml and E. faecalis 16
mg/ml. The MIC o f A. recurvifolia is 8 mg/ml for E. coli, 8 mg/ml for S. aureus and
16 mg/ml for E. faecalis. Well 10E on the microplate showed no INT colouring. This
can be attributed to the fact that well 10E was not inoculated with culture (E. coli).
The MIC for A. recurvifolia for E. coli however stays 8 mg/ml.
The bacteriostatic and fungistatic activities o f the essential oils were evaluated by
using the undiluted oils o f the Agathosma species in the screening tests. Quantitative
results were determined by calculating the minimum inhibitory concentration (MIC),
using the serial dilution method. Agathosma ovata (Gamka) had zones o f inhibition
o f less than 1.0 mm on E. coli, 5.0 mm on E. faecalis and less than 1.0 mm on S.
aureus. The final values taken after 24 hours MIC, for A. ovata (Gamka) on the same
bacteria were 16 mg/ml, 16 mg/ml and 8 mg/ml. Agathosma ovata (Anysberg) had
56
zones o f inhibition o f 3.0 mm on E. coli, 2.0 mm on E. faecalis and 1.0 mm on S.
aureus. The MIC for A. ovata (Anysberg) on the same bacteria were 8 mg/ml, 16
mg/ml and 8 mg/ml. Agathosma capensis (Gamka) had zones o f inhibition o f 1.0 mm
on E. coli, 3.0 mm on E. faecalis and 2.0 mm on S. aureus. The M IC’s for A.
capensis (Gamka) on the same bacteria were 16 mg/ml, 32 mg/ml and 32 mg/ml
respectively. Agathosma recurvifolia had zones o f inhibition o f 1.0 mm on E. coli,
7.0 mm on E. faecalis and 1.5 mm on S. aureus. The MIC for A. recurvifolia on the
same bacteria was 8 mg/ml, 16 mg/ml and 8 mg/ml respectively. The MIC results
therefore correlate with what was observed in the disc diffusion screening results.
The TLC bioautographic assay (Figure 40) with the hydrodistilled oil o f A.
zwartbergensis showed one zone o f inhibition. A TLC bioautographic assay o f pure
citronellal was done simultaneously with the assay o f the hydrodistilled oil. As
indicated on figure 40, the main compound o f A. zwartbergensis, the yellow
compound (R f = 0.79) on the TLC vanillin-sulphuric plate, correlates with the
citronellal standard. This compound was also identified with GC-MS as being
citronellal and could be the antimicrobial factor o f the essential oil.
57
T ab le 11: A n tib a cter ia l sc r e e n in g resu lts as e x p r e sse d in th e d isc d if fu s io n a ssa y (m m from d isc e d g e ).
S p e c ie s E scherich ia co li E nterococcus
faecalis
P seudom onas
aeruginosa
Salm onella
typhim urium
B acillus cereus Staphylococcus
aureus
N e o m y c in 3 0 p g , (O x o id ) 5 .0 3 .0 2 .0 4 .0 10 .0 1 0 .0
A. arida R * R * R * < 1 . 0 2 .0 R *
A. capensis (G a m k a ) 1 .0 3 .0 R* 1.0 1.0 2 .0
A. capensis (R o o ib e r g ) 1 .0 R * R* 1.0 R * 1.5
A. capensis (M o s e l B a y ) < 1 .0 2 .0 R* R * R * 2 .0
A. lanata R * R * R* R * 1.0 R*
A. m undtii R * 1.0 R* < 1 .0 R * 1.0
A. ovalifo lia R * 2 .0 R* R * R * < 1 .0
A. ovata (G a m k a ) < 1 .0 5 .0 R* < 1 .0 2 .0 < 1 .0
A. ovata ( A n y s b e rg ) 3 .0 2 .0 1.0 < 1 . 0 1.0 1.0
A. recurvifo lia 1.0 7 .0 R * R * R* 1.5
A. serpyllacea < 1 .0 3 .0 R * < 1 .0 2 .0 2 .5
A. zw artbergensis R * R * R* R * 3 .0 R *
*R = Resistant
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Figure 37: Disc diffusion plate o f E. coli on the essential oils o f Agathosma species studied.
Figure 38: Disc diffusion plate o f B. cereus on essential oils o f (34 - A capensis (Gamka); 3 6 - A. ovata (Gamka); 38 — A. zwartbergensis; 40 -A . ovalifolia).
i ( 9 1 • • - # * • ; #
i * t * ' •' «r*» * or m,*.- *;**,'*,
Figure 39: MIC results after 24 hours.
Figure 40: TLC bioautographic assay.
Table 12: Antifungal disc diffusion screening results (expressed as mm from disc edge).
Species C andida alb icans C ryp tococcus neo form ans A sp erg illu s n iger
Nystatin (100 iu, Oxoid). 7.0 5.0 7.0
A. arida R* 3.0 R*
A. capensis (Gamka) R* < 1.0 R*
A. capensis (Rooiberg) R* < 1.0 R*
A. capensis (Mossel Bay) R* 2.0 R*
A. lanata R* 2.0 R*
A. m undtii R* <1.0 R*
A. ova lifo lia 2.0 1.0 1.0
A. ovata (Gamka) 1.0 2.0 R*
A. ovata (Anysberg) R* 2.0 R*
A. recurv ifo lia 2.0 2.0 R*
A. serpyllacea 2.0 2.0 R*
A. zw artbergensis 3.0 2.0 R*
*R = Resistant
60
Table 13: MIC results after 30 minutes, 2 hours and 24 hours.
Microplate
column
Test organism Species MIC (mg/ml) after 30
min
MIC (mg/ml) after 2 hours MIC (mg/ml) after 24
hours
1 E schericha co li A. capensis (Gamka) 8 8 16
2 S taphylococcus aureus A. capensis (Gamka) 8 16 32
3 E nterococcus fa e c a lis A. capensis (Gamka) No colouring 16 32
4 E schericha coli A. ovata (Gamka) 4 8 • 16
5 Staphylococcus aureus A. ovata (Gamka) 4 4 8
6 E nterococcus fa e c a lis A. ovata (Gamka) 2 8 16
7 E schericha co li A. ovata (Anysberg) 4 4 8
8 Staphylococcus aureus A. ovata (Anysberg) 2 4 8
9 E nterococcus fa e c a lis A. ovata (Anysberg) 2 8 16
10 E schericha co li A. recurvifo lia 4 4 8
11 S taphylococcus aureus A. recurvifo lia 2 4 8
12 E nterococcus fa e c a lis A. recurvifo lia 1 8 16
61
4.2 Analytical chemistry
TLC plates of all 10 essential oils were developed and detection was made possible with
the use o f spray-reagents namely vanillin-sulphuric acid (Figure 41) and anisaldehyde-
sulphuric acid (Figure 42). For the TLC plate where vanillin-sulphuric acid was used, the
following similarities were seen on the TLC plates of the individual essential oils.
Agathosma mundtii and A. ovalifolia have similar compounds (coloured blue after
development of the plate) with R f = 0.90. There is a consistency between all 10 samples
in the middle region of the TLC plate. This indicates similar compounds in the different
essential oils. Agathosma capensis (Gamka) contains a unique compound (coloured
brown after development of the plate) with R f = 0.90. Agathosma zwartbergensis
contains a unique compound (coloured yellow-brown after development) with R f = 0.79.
A. ovalifolia contains a unique compound (colour yellow on TLC plate) with R f = 0.59
and Agathosma species contains a unique compound (colour pink) with R f = 0.52.
Similar results were obtained with the anisaldehyde-sulphuric acid colour reagent. Thin
layer chromatography is however not very useful when working with complex mixtures
such as essential oils. The purpose of this study was merely to also produce a fingerprint
of the species studied (e.g. A. zwartbergensis) and to visually summarize the immense
variation between the selected species.
The GC-MS results are tabulated under the monographs o f each species and the major
compounds are clearly indicated. Three samples o f A. capensis were analyzed by GC-
MS. The one sample was harvested from the Gamka Mountains, the other sample was
harvested from the Rooiberg region in the Cape Province and the last sample was
collected from Mossel Bay. There are very interesting differences between the
composition o f the three samples and these results support the fact that external factors
may influence the chemical compositions of species. All three samples had the following
same major compounds: linalool, myrcene and limonene. Methyl-chavicol is a major
compound o f A. capensis (Gamka) but not for A. capensis (Rooiberg) and A. capensis
(Mossel Bay). Sabinene is a major compound for A. capensis (Rooiberg) but not for A.
capensis (Gamka) and A. capensis (Mossel Bay). /3-phellandrene, (Z)-/3-ocimene and
62
Figure 41: TLC plate o f Agathosma essential oils. The plate has been treated with vanillin-sulphuric acid.
#
i i * I * i i I * * «
Figure 42: TLC plate o f Agathosma essential oils. The plate has been treated with anisaldehyde-sulphuric acid.
(E)-P-ocimene are major compounds for A. capensis (Mossel Bay) but not for A. capensis
(Gamka) and A. capensis (Rooiberg). It is important to note that one of the major
compounds of A. mundtii with the highest percentage area (20.00%) could not be
identified using GC-MS. This compound had a base peak of 69 and a molecular mass of
172. This compound could not be suitably matched on many GC-MS libraries and
judging by the comprehensive database used it could be a possible new compound.
Two samples of A. ovata were analyzed by GC-MS where the one sample was collected
from the Gamka Mountains and the other sample was collected form the Anysberg region
in the Cape. Both samples contained sabinene and terpinen-4-ol as major compounds.
There were however some differences in the compositions of the two samples that may
be attributed to external factors. Myrcene and linalool are major compounds of A. ovata
(Gamka) but not for A. ovata (Anysberg). Limonene, p-cymene and p-phellandrene are
major compounds of A. ovata (Anysberg) but not for A. ovata (Gamka).
Previous analytical chemistry research has been done on commercial Agathosma species.
These species include A. betulina and A. crenulata. The major compounds in the
essential oil of A. betulina are isomenthone and diosphenol. Other compounds identified
in A. betulina include limonene, menthone, pulegone, terpinen-4-ol and p-menthan-3-on-
8-thiol. Agathosma crenulata contains a less desirable compound namely pulegone (van
Wyk and Gericke, 2000; Bisset, 1994). None of the Agathosma species in this study
contained isomenthone, pulegone, diosphenol, menthone or />menthan-3-on-8-thiol. All
the Agathosma species in this study contained limonene that was found in previous
studies of A. betulina and A. crenulata. All the species studied except the A. capensis
(Gamka) sample contained terpinen-4-ol, which is also found in A. betulina and A.
crenulata.
64
4.3 Antimicrobial activity of main compoundsAll the Agathosma species studied showed some degree of antimicrobial activity.
Literature studied on essential oil containing plant species show similarities in
antimicrobial activity with the compounds found in some of the Agathosma species.
A study was done by Carson and Riley (1995) to examine the antimicrobial activity of
eight individual components of Tea Tree oil. Tea Tree oil is commonly used to treat skin
disorders such as cuts, bums, insect bites and athlete’s foot. Tea Tree oil contains 1,8-
cineole, l-terpinen-4-ol, p-cymene, linalool, a-terpinene, y-terpinene, a-terpineol and
terpinolene. The test organisms included Candida albicans, Enterococcus faecalis,
Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. The results of
the study indicated that terpinen-4-ol was active against all test organisms. Linalool and
a-terpineol were active against all the tested organisms except Pseudomonas aeruginosa.
In this study the Agathosma species containing linalool and a-terpineol as main
compounds were also active against all the test organisms except Pseudomonas
aeruginosa. Agathosma mundtii and A. ovata (Gamka and Anysberg) contain terpinen-4-
ol as a main compound. Agathosma ovata (Anysberg) showed activity towards all the
test organisms, A. mundtii showed activity towards all test organisms except E. coli and
P. aeruginosa and A. ovata (Gamka) showed activity towards all test organisms except P.
aeruginosa. Terpinen-4-ol, p-cymene, linalool and a-terpineol are present, not necessarily
as major components, in all the Agathosma species studied.
Cimanga et al. (2002) indicates that Eucalyptus, Aframomum, Ocimum, Cympobogon and
Monodora species, from the Democratic Republic o f the Congo, contain the essential oil
compounds 1,8-cineole, a-pinene and P-pinene, p-cymene, myrcene, y-terpinene, a-
terpineol, limonene, P-terpineol, citronellal, cryptone, phellandrene and thymol. The
results published in the article indicated that these essential oils showed inhibition of
selected bacterial growth. They compared the chemical composition of the essential oils
of Eucalyptus camadulensis and Cympobogon citratus with their antibacterial activity
and found that their activity is related to the high levels of 1,8-cineole, geranial and neral.
65
Similar bacteria were tested in this study of the antimicrobial activity of buchu and
include E. coli, P. aeruginosa and S. aureus. The Agathosma species studied contain
similar essential oil compounds as the plant species from the Congo. All the Agathosma
species studied contain a-pinene, /3-pinene, p-cymene, myrcene, a-terpineol and limonene
as major or minor compounds.
The essential oil o f Phlomis lanata contains the major compounds; a-pinene, limonene
and trans-caryophyllene. Couladis et al. (2000) on the antimicrobial activity and
chemical composition of Phlomis lanata, indicates that the oil had moderate activity
against the bacteria tested and strong activity against the test fungi. Pure limonene and a-
pinene were tested on the same cultures and the results in the article suggest that the
activity of the oil could be largely attributed to these two main compounds o f the oil.
Agathosma capensis (Gamka and Rooiberg), A. ovata (Anysberg), A. recurvifolia and A.
ovalifolia contain limonene as a major compound. Agathosma mundtii, A. ovata (Gamka)
and A. recurvifolia contain a-pinene as a major compound. The essential oils o f these
Agathosma species with the same major compounds as the essential oil o f Phlomis lanata
showed similar activity towards similar test organisms (E. coli, S. aureus, P. aeruginosa
and C. albicans). Agathosma arida also contains limonene and a-pinene but did not
show activity towards these test organisms. It is noteworthy that all the buchu species
studied contain limonene and a-pinene either as a minor or a major compound.
Cobos et al. (2001) investigated the chemical composition and antimicrobial activity of
the essential oil of Baccharis notosergila. The major compounds were a-pinene,
limonene, j8-caryophyllene and spathulenol. They came to the conclusion that essential
oils containing monoterpenes like limonene are more active against gram-positive
organisms and fungi than gram-negative organisms. Agathosma capensis (Gamka and
Rooiberg), A. ovata (Anysberg), A. recurvifolia and A. ovalifolia contain limonene, a
monoterpene, as a major compound and showed activity towards the gram-positive
bacteria Enterococcus faecalis, Bacillus cereus and Staphylococcus aureus.
The antibacterial activity of Eucalyptus essential oils is due to the synergy of citronellol
and citronellal (Zakarya et al, 1993). Agathosma zwartbergensis contains citronellal as a
66
main constituent and citronellol as a minor constituent. Agathosma mundtii, A. capensis
(Gamka and Rooiberg), A. ovata (Anysberg), A. recurvifolia and A. arida contain
citronellol as a minor constituent. Agathosma ovata (Gamka) contains citronellol and
citronellal as minor constituents.
The positioning of functional groups (terpinen-4-ol compared to oterpineol), level of
ring saturation (carvone compared to dihydrocarvone), type of functional group present
and the level o f chain saturation in an acyclic terpenoid (geraniol compared to citronellol)
determine the antibacterial activity of monoterpenes. Small changes in molecular
properties affect the permeation through bacterial outer membranes and therefore the
antibacterial activity (Griffin et al., 2001). Griffin et al. (1999) examined the structure-
activity relationships of terpenoids. Low water solubility, mainly essential oil
compounds containing hyrocarbons and acetates, attribute to the relative inactivity of
essential oils. Furthermore the antimicrobial activity of oxygenated terpenoid containing
essential oils is associated with hydrogen bonding. It is important to note that water
solubility and hydrogen bonding does not account for all the trends in the activity of
essential oils. The activity o f geraniol, nerol and linalool is largely determined by the
presence o f the alcohol functional group on the carbon skeleton o f these acyclic
terpenoids. The hydrogen-bonding capacity and hence activity is illustrated when
comparing the activity of citronellol, inactive towards E. coli, and geraniol, active
towards E. coli. Citronellal, the corresponding aldehyde to citronellol, was also inactive
towards E. coli and can be attributed to the lower solubility (less hydrogen bonding) than
geraniol, linalol and nerol (Griffin et al., 1999).
When comparing the main constituents o f the Agathosma species studied with the main
constituents o f other species showing antibacterial activity, the compounds present in the
greatest proportions are not necessarily responsible for the greatest share o f the total
activity. It is important to consider the less abundant constituents and the possibility of
synergy between components.
67
5. Conclusion
The main objectives of this study were to investigate the possible antimicrobial properties
of a selection of species belonging to the genus Agathosma and to record the essential oil
profiles of these species.
The presence of antibacterial and antifungal activity of the various Agathosma species
were proven in this study. The activity varied amongst the studied species. Agathosma
recurvifolia for E. faecalis had an activity greater than the positive control (Neomycin 30
fig (Oxoid) used. After comparing the results with antimicrobial results o f other essential
oil containing plant species, it was noted that it is important to consider the less abundant
compounds of the essential oils. The compounds in the greatest proportion are not
necessarily responsible for the antimicrobial activity and the possibility o f synergy
between compounds should be considered. A TLC bioautographic assay was conducted
to determine the antimicobial factor o f A. zwarbergensis. Results indicated that
citronellal could possibly be responsible for the observed antimicrobial activity.
The twelve oils were subjected to GC-MS and the profiles were recorded. For all twelve
oils more than 90% of the compounds were identified. However the main compound of
A. mundtii could not be identified and is most probably a new terpenoid. Each species
still has a unique qualitative and quantitative composition. The differences amongst and
within species could possibly be attributed to some external factors that include the
botanical source, the condition of the plant material (fresh or dried) and the isolation
technique (steam distillation or hydrodistillation).
The results of this report can support the use o f these medicinal plants, generally referred
to as ‘Buchu’, as traditional remedies for selected infectious diseases.
68
6. References
B a ra tta M ., D o r m a n H ., D e a n s S ., F ig u e ir e d o A ., B a r ro so J. and R u b e rto G . 1 9 9 8 . A n t im ic r o b ia l and
a n tio x id a n t p r o p e r tie s o f s o m e c o m m e r c ia l e s s e n t ia l o i ls . F la v o u r an d F ra g ra n ce Journal 13: 2 3 5 -2 4 4 .
<r
“iB is s e t N .G . e d ito r . 1 9 9 4 . H erb a l D r u g s and P h y to p h a r m a c e u tic a ls . L o n d o n : C R C P re ss .
B u c h b a u e r G . 1 9 9 3 . B io lo g ic a l e f fe c t s o f fra g ra n ces and e s se n t ia l o i ls . P er fu m er an d F la v o r is t 18: 1 9 -2 4 .
C a r so n C . a n d R ile y T . 1 9 9 5 . A n tim ic r o b ia l a c t iv ity o f th e m ajor c o m p o n e n ts o f th e e s se n t ia l o i l o f
M ela leuca a ltern ifo lia . Jou rn al o f A p p lie d B a c te r io lo g y 78 : 2 6 4 -2 6 9 . <'
C im a n g a K ., K a m b u K., T o n a L ., A p er s S ., D e B r u y n e T., H erm a n s N., e t al. 2 0 0 2 . C o rre la tio n b e tw e e n
c h e m ic a l c o m p o s it io n and a n tib a c ter ia l a c t iv ity o f e s s e n t ia l o i ls o f so m e a ro m a tic m e d ic in a l p lan ts
g r o w in g in th e D e m o c r a t ic R e p u b lic o f C o n g o . Journal o f E th n o p h a r m a c o lo g y 7 9 : 2 1 3 -2 2 0 .
C o b o s M ., R o d r ig u e z J., O liv a M ., D e m o M ., F a il la c i S . an d Z y g a d lo J. 2 0 0 1 . C o m p o s it io n and
a n tim ic r o b ia l a c t iv ity o f th e e s se n t ia l o i l o f B accharis notosergila . P la n ta M e d ic a 67: 8 4 -8 6 .
C o ll in s N . , G ra v en E ., v a n B e e k T . and L e ly v e ld G . 1 9 9 6 . C h e m o ta x o n o m y o f c o m m e r c ia l B u c h u s p e c ie s
(A gathosm a be tu lina a n d A g a th o sm a crenula ta). Jou rn al o f E s s e n t ia l O il R e se a r c h 8: 2 2 9 - 2 3 5 .
C o m b e s t W . T e a tree . 2 0 0 0 . T h e Jou rn al o f M o d e rn P h a r m a cy 2 0 -2 2 . ' -
C o u la d is M ., T a n im a n id is A ., T z a k o u O ., C h in o u 1. an d H a rv a la C . 2 0 0 0 . E sse n tia l o i l o f P hlom is lana ta
g r o w in g in G reece : C h e m ic a l c o m p o s it io n an d a n tim ic r o b ia l a c tiv ity . P la n ta M e d ic a 6 6 : 6 7 0 - 6 7 1 .
C o w a n M .M . 1 9 9 9 . P la n t p r o d u cts a s a n tim ic r o b ia l a g e n ts . C lin ic a l M ic r o b io lo g ic a l R e v ie w s 12: 5 6 4 -5 8 2 .
E v a n s W . 1 9 9 6 . T r e a se an d E v a n s P h a r m a c o g n o sy . 1 4 th e d it io n . W .B S a u n d ers C o m p a n y L td ., L o n d o n .
F la m in i G ., C io n i P ., P u le io R ., M o r e ll i I. an d P a n iz z i L . 1 9 9 9 . A n tim ic r o b ia l a c t iv ity o f th e e s se n t ia l o i l o f
C alam in tha nepeta a n d its c o n s t itu e n t p u le g o n e a g a in s t b a c te r ia an d fu n g i. P h y to th e r a p y R e se a r c h 13:
3 4 9 - 3 5 1 .
69
G riff in S ., W y llie S ., M ark h am J. an d L ea ch D . 1 9 9 9 . T h e r o le o f structure an d m o le c u la r p r o p e r tie s o f
te r p e n o id s in d e te r m in in g th e ir a n tim icro b ia l a c t iv ity . F la v o u r an d F ragran ce Jou rn al 14: 3 2 2 -3 3 2 .
G r iff in S ., W y llie S . and M ark h am J. 2 0 0 1 . R o le o f th e o u ter m e m b ra n e o f E sch eric ia co li A G 1 0 0 and
P seudom onas aerug inosa N C T C 6 7 4 9 a n d r e s is ta n c e /su sc e p tib il ity to m o n o te r p e n e s o f s im ila r
c h e m ic a l stru c tu re . Journal o f E sse n tia l O il R e se a r c h 13: 3 8 0 -3 8 6 .
G o ld b la tt P . an d M a n n in g J. 2 0 0 0 . C a p e P la n ts. A c o n sp e c tu s o f th e C a p e F lo r a o f S o u th A fr ic a . N a tio n a l
B o ta n ic a l In st itu te o f S o u th A fr ica . P retoria .
Iw u M ., D u n c a n A . an d O k u n ji C . 1 9 9 9 . N e w a n t im ic r o b ia ls o f p la n t o r ig in . P e r s p e c t iv e s o n N e w C rop s
an d N e w U s e s . J. J a n ick (E d ), A S H S P re ss , A le x a n d r ia , V A .
J a n sse n A ., S c h e f fe r J. an d B a e rh e im S v e n d se A . 1 9 8 6 . A n tim ic r o b ia l a c t iv ity o f e s s e n t ia l o ils : A 1 9 7 6 -
1 9 8 6 litera tu re r e v ie w . A s p e c ts o f th e te s t m e th o d s . P la n ta M e d ic a 53: 3 9 5 -3 9 8 .
K a ise r R ., L a m p a r sk y D . and S c h u d e l P . 1 9 7 5 . A n a ly s is o f b u ch u l e a f o il . Journal o f A g r icu ltu ra l and
F o o d C h e m istr y 2 3 : 9 4 3 -9 5 0 .
K h a llo u k i F ., H m a m o u c h i M ., Y o u n o s C ., S o u lim a n i R ., B e s s ie r e J. an d E s s a s s i E . 2 0 0 0 . A n tib a c te r ia l and
m o llu s c ic id a l a c t iv it ie s o f th e e s se n t ia l o il o f C hrysanthem um viscidehirtum . F ito te r a p ia 7 1 : 5 4 4 -5 4 6 .
L a w r e n c e B . 1 9 7 6 . R e c e n t p r o g r ess in e sse n tia l o ils : b u ch u o i l . P e r fu m e r an d F la v o r is t 1 : 1 7 ,
M a n g e n a T . an d M u y im a N . 1 9 9 9 . C o m p a ra tiv e e v a lu a t io n o f th e a n tim ic r o b ia l a c t iv it ie s o f e s s e n t ia l o i ls
o f A rtim is ia afra, P teronia incana an d R osm arinus o ffic ina lis o n s e le c te d b a c te r ia a n d y e a s t stra in s.
L e tter s in A p p lie d M ic r o b io lo g y 2 8 : 2 9 1 -2 9 6 . C f , 1' ' ?
N a k a tsu T ., L u p o A ., C h in n J. and K a n g R . 2 0 0 0 . B io lo g ic a l a c t iv ity o f e s s e n t ia l o i l s a n d th e ir c o n s t itu e n ts .
S tu d ie s in N a tu ra l P ro d u cts C h e m istr y V o l 2 1 . A tta -u r-R a h m a n (E d ), E ls e v ie r S c ie n c e s B .V .
P o s th u m u s M ., v a n B e e k T ., C o ll in s N . an d G ra v en E. 1 9 9 6 . C h e m ic a l c o m p o s it io n o f t h e e s se n t ia l o i ls o f
A g a th o sm a betulina, A. crenu la ta a n d an A. betu lina x crenu la ta h yb rid (b u c h u ) . Jou rn a l o f E sse n tia l
O il R e se a r c h 8: 2 2 3 - 2 2 8 .
T z a k o u O ., P ita r o k ili D ., C h in o u I. an d H arva la C . 2 0 0 1 . C o m p o s it io n and a n tim ic r o b ia l a c t iv ity o f the
e s se n t ia l o i l o f S a lv ia ringens. P la n ta M e d ic a 6 7 : 8 1 -8 3 .
70
v a n W y k B -E ., v a n O u d tsh o o r n B . and G er ick e N . 1 9 9 7 . M e d ic in a l P la n ts o f S o u th A fr ic a . B r iz a , P retoria ,
v a n W y k B -E . and G e r ic k e N . 2 0 0 0 . P e o p le ’s P la n ts . B r iz a , P reto ria .
W a tt J. a n d B r e y e r -B r a n d w ijk M . 1 9 6 2 . T h e M e d ic in a l and P o is o n o u s P la n ts o f S o u th e rn an d E astern
A fr ic a . S e c o n d e d it io n . L o n d o n : E and S L iv in g s to n e .
Z ak arya D . , F k ih -T e to u a n i S . and H ajji F. 1 9 9 3 . C h e m ic a l c o m p o s it io n -a n t im ic r o b ia l a c t iv ity r e la tio n sh ip
o f E u ca lyp tu s e s s e n t ia l o i ls . P la n te s M e d ic in a le s e t P h y to th e r a p ie 2 6 : 3 3 1 -3 3 9 .
h ttp ://w w w .n a tu r a la r o m a th e r a p y .c o m /a r 0 2 .h tm ( A c c e s s e d 0 9 .0 8 .2 0 0 2 )
h ttp : //w w w .o ilso f i ia tu r e .c o m .a u /T e a _ T r e e _ O il/h is to r y _ o f_ te a _ tr e e _ o iI .h tm ( A c c e s s e d 0 9 .0 8 .2 0 0 2 )